Methods for delivering DNA to muscle cells using recombinant adeno-associated virus virions

ABSTRACT

The use of recombinant adeno-associated virus (AAV) virions for delivery of DNA molecules to muscle cells and tissue is disclosed. The invention allows for the direct, in vivo injection of recombinant AAV virions into muscle tissue, e.g., by intramuscular injection, as well as for the in vitro transduction of muscle cells which can subsequently be introduced into a subject for treatment. The invention provides for sustained, high-level expression of the delivered gene and for in vivo secretion of the therapeutic protein from transduced muscle cells such that systemic delivery is achieved.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 08/588,355, filed Jan. 18, 1996, from which priority is claimedpursuant to 35 USC §120 and which is incorporated herein by reference inits entirety.

TECHNICAL FIELD

[0002] The present invention relates generally to DNA delivery methods.More particularly, the invention relates to the use of recombinantadeno-associated virus (AAV) virions for delivery of a selected gene tomuscle cells and tissue. The method provides for sustained, high-levelexpression of the delivered gene.

BACKGROUND OF THE INVENTION

[0003] Gene delivery is a promising method for the treatment of acquiredand inherited diseases. Muscle tissue is an appealing gene deliverytarget-because it is readily accessible, well-differentiated andnondividing. Barr and Leiden (1991) Science 254:1507-1509. Theseproperties are important in the selection of appropriate deliverystrategies to achieve maximal gene transfer.

[0004] Several experimenters have demonstrated the ability to delivergenes to muscle cells with the subsequent systemic circulation ofproteins encoded by the delivered genes. See, e.g., Wolff et al. (1990)Science 247:1465-1468; Acsadi et al. (1991) Nature 352:815-818; Barr andLeiden (1991) Science 254:1507-1509; Dhawan et al. (1991) Science254:1509-1512; Wolff et al. (1992) Human Mol. Genet. 1:363-369; Eyal etal. (1993) Proc. Natl. Acad. Sci. USA 90:4523-4527; Davis et al. (1993)Hum. Gene Therapy 4:151-159.

[0005] Genes have been delivered to muscle by direct injection ofplasmid DNA, such as described by Wolff et al. (1990) Science247:1465-1468; Acsadi et al. (1991) Nature 352:815-818; Barr and Leiden(1991) Science 254:1507-1509. However, this mode of administrationgenerally results in sustained but low levels of expression. Low butsustained expression levels may be effective in certain situations, suchas for providing immunity.

[0006] Viral based systems have also been used for gene delivery tomuscle. For example, human adenoviruses are double-stranded DNA viruseswhich enter cells by receptor-mediated endocytosis. These viruses havebeen considered well suited for gene transfer because they are easy togrow and manipulate and they exhibit a broad host range in vivo and invitro. Adenoviruses are able to infect quiescent as well as replicatingtarget cells and persist extrachromosomally, rather than integratinginto the host genome.

[0007] Despite these advantages, adenovirus vectors suffer from severaldrawbacks which make them ineffective for long term gene therapy. Inparticular, adenovirus vectors express viral proteins that may elicit animmune response which may decrease the life of the transduced cell. Thisimmune reaction may preclude subsequent treatments because of humoraland/or T cell responses. Furthermore, the adult muscle cell may lack thereceptor which recognizes adenovirus vectors, precluding efficienttransduction of this cell type using such vectors. Thus, attempts to useadenoviral vectors for the delivery of genes to muscle cells hasresulted in poor and/or transitory expression. See, e.g., Quantin et al.(1992) Proc. Natl. Acad. Sci. USA 89:2581-2584; Acsadi et al. (1994)Hum. Mol. Genetics 3:579-584; Acsadi et al. (1994) Gene Therapy1:338-340; Dai et al. (1995) Proc. Natl. Acad. Sci. USA 92:1401-1405;Descamps et al. (1995) Gene Therapy 2:411-417; Gilgenkrantz et al.(1995) Hum. Gene Therapy 6:1265-1274.

[0008] Gene therapy methods based upon surgical transplantation ofmyoblasts has also been attempted. See, e.g., International Publicationno. WO 95/13376; Dhawan et al. (1991) Science 254:1509-1512; Wolff etal. (1992) Human Mol. Genet. 1:363-369; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hamamori et al. (1994) Hum. Gene Therapy5:1349-1356; Hamamori et al. (1995) J. Clin. Invest. 95:1808-1813; Blauand Springer (1995) New Eng. J. Med. 333:1204-1207; Leiden, J. M. (1995)New Eng. J. Med. 333:871-872; Mendell et al. (1995) New Eng. J. Med.333:832-838; and Blau and Springer (1995) New Eng. J. Med.333:1554-1556. However, such methods require substantial tissue culturemanipulation and surgical expertise, and, at best, show inconclusiveefficacy in clinical trials. Thus, a simple and effective method of genedelivery to muscle, resulting in long-term expression of the deliveredgene, would be desirable.

[0009] Recombinant vectors derived from an adeno-associated virus (AAV)have been used for gene delivery. AAV is a helper-dependent DNAparvovirus which belongs to the genus Dependovirus. AAV requiresinfection with an unrelated helper virus, such as adenovirus, aherpesvirus or vaccinia, in order for a productive infection to occur.The helper virus supplies accessory functions that are necessary formost steps in AAV replication. In the absence of such infection, AAVestablishes a latent state by insertion of its genome into a host cellchromosome. Subsequent infection by a helper virus rescues theintegrated copy which can then replicate to produce infectious viralprogeny. AAV has a wide host range and is able to replicate in cellsfrom any species so long as there is also a successful infection of suchcells with a suitable helper virus. Thus, for example, human AAV willreplicate in canine cells coinfected with a canine adenovirus. AAV hasnot been associated with any human or animal disease and does not appearto alter the biological properties of the host cell upon integration.For a review of AAV, see, e.g., Berns and Bohenzky (1987) Advances inVirus Research (Academic Press, Inc.) 32:243-307.

[0010] The AAV genome is composed of a linear, single-stranded DNAmolecule which contains approximately 4681 bases (Berns and Bohenzky,supra). The genome includes inverted terminal repeats (ITRS) at each endwhich function in cis as origins of DNA replication and as packagingsignals for the virus. The internal nonrepeated portion of the genomeincludes two large open reading frames, known as the AAV rep and capregions, respectively. These regions code for the viral proteinsinvolved in replication and packaging of the virion. For a detaileddescription of the AAV genome, see, e.g., Muzyczka, N. (1992) CurrentTopics in Microbiol. and Immunol. 158:97-129.

[0011] The construction of recombinant AAV (rAAV) virions has beendescribed. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941;International Publication Numbers WO 92/01070 (published Jan. 23, 1992)and WO 93/03769 (published Mar. 4, 1993); Lebkowski et al. (1988) Molec.Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold SpringHarbor Laboratory Press); Carter, B. J. (1992) Current Opinion inBiotechnology 3:533-539; Muzyczka, N. (1992) Current Topics inMicrobiol. and Immunol. 158:97-129; and Kotin, R. M. (1994) Human GeneTherapy 5:793-801.

[0012] Recombinant AAV virion production generally involvescotransfection of a producer cell with an AAV vector plasmid and ahelper construct which provides AAV helper functions to complementfunctions missing from the AAV vector plasmid. In this manner, theproducer cell is capable of expressing the AAV proteins necessary forAAV replication and packaging. The AAv vector plasmid will include theDNA of interest flanked by AAV ITRs which provide for AAV replicationand packaging functions. AAV helper functions can be provided via an AAVhelper plasmid that includes the AAV rep and/or cap coding regions butwhich lacks the AAV ITRs. Accordingly, the helper plasmid can neitherreplicate nor package itself.

[0013] The producer cell is then infected with a helper virus to provideaccessory functions, or with a vector which includes the necessaryaccessory functions. The helper virus transactivates the AAV promoterspresent on the helper plasmid that direct the transcription andtranslation of AAV rep and cap regions. Upon subsequent culture of theproducer cells, recombinant AAV virions harboring the DNA of interest,are produced.

[0014] Recombinant AAV virions have been shown to exhibit tropism forrespiratory epithelial cells (Flotte et al. (1992) Am. J. Respir. CellMol. Biol. 7:349-356; Flotte et al. (1993) J. Biol. Chem. 268:3781-3790;Flotte et al. (1993) Proc. Natl. Acad. Sci. USA 90:10613-10617) andneurons of the central nervous system (Kaplitt et al. (1994) NatureGenetics 8:148-154). These cell types are well-differentiated,slowly-dividing or postmitotic. Flotte et al. (1993) Proc. Natl. Acad.Sci. USA 90:10613-10617; Kaplitt et al. (1994) Nature Genetics8:148-154. The ability of AAV vectors to transduce nonproliferatingcells (Podsakoff et al. (1994) J. Virol. 68:5656-5666; Russell et al.(1994) Proc. Natl. Acad. Sci. USA 91:8915-8919; Flotte et al. (1994) Am.J. Respir. Cell Mol. Biol. 11:517-521) along with the attributes ofbeing inherently defective and nonpathogenic, place AAV in a uniqueposition among gene therapy viral vectors.

[0015] Despite these advantages, the use of recombinant AAV virions todeliver genes to muscle cells in vivo has not heretofore been disclosed.

SUMMARY OF THE INVENTION

[0016] Accordingly, the present invention is based on the surprising andunexpected discovery that recombinant. AAV (rAAV) virions provide forefficient delivery of genes and sustained production of therapeuticproteins in various muscle cell types. The invention allows for in vivosecretion of the therapeutic protein from transduced muscle cells suchthat systemic delivery of therapeutic levels of the protein is achieved.These results are seen with both in vivo and in vitro modes of DNAdelivery. Hence, rAAV virions allow delivery of DNA directly to muscletissue. The ability to deliver and express genes in muscle cells, aswell as to provide for secretion of the produced protein from transducedcells, allows the use of gene therapy approaches to treat and/or preventa wide variety of disorders.

[0017] Furthermore, the ability to deliver DNA to muscle cells byintramuscular administration in vivo provides a more efficient andconvenient method of gene transfer.

[0018] Thus, in one embodiment, the invention relates to a method ofdelivering a selected gene to a muscle cell or tissue. The methodcomprises:

[0019] (a) providing a recombinant AAV virion which comprises an AAVvector, the AAV vector comprising the selected gene operably linked tocontrol elements capable of directing the in vivo transcription andtranslation of the selected gene; and

[0020] (b) introducing the recombinant AAV virion into the muscle cellor tissue.

[0021] In particularly preferred embodiments, the selected gene encodesa therapeutic protein, such as erythropoietin (EPO), or the lysosomalenzyme, acid α-glucosidase (GAA).

[0022] In another embodiment, the invention is directed to a muscle cellor tissue transduced with a recombinant AAV virion which comprises anAAV vector, the AAV vector comprising a selected gene operably linked tocontrol elements capable of directing the in vivo transcription andtranslation of the selected gene.

[0023] In still further embodiments, the invention is directed to amethod of treating an acquired or inherited disease in a mammaliansubject comprising introducing into a muscle cell or tissue of thesubject, in vivo, a therapeutically effective amount of a pharmaceuticalcomposition which comprises (a) a pharmaceutically acceptable excipient;and (b) recombinant AAV virions.

[0024] The recombinant AAV virions comprise an AAV vector, the AAVvector comprising a selected gene operably linked to control elementscapable of directing the transcription and translation of the selectedgene when present in the subject.

[0025] In yet another embodiment, the invention is directed to a methodof treating an acquired or inherited disease in a mammalian subjectcomprising:

[0026] (a) introducing a recombinant AAV virion into a muscle cell ortissue in vitro to produce a transduced muscle cell. The recombinant AAVvirion comprises an AAV vector, the AAV vector comprising a selectedgene operably linked to control elements capable of directing thetranscription and translation of the selected gene when present in thesubject; and

[0027] (b) administering to the subject a therapeutically effectiveamount of a composition comprising a pharmaceutically acceptableexcipient and the transduced muscle cells from step (a).

[0028] In a further embodiment, the invention relates to a method fordelivering a therapeutically effective amount of a protein systemicallyto a mammalian subject comprising introducing into a muscle cell ortissue of the subject a pharmaceutical composition which comprises (a) apharmaceutically acceptable excipient; and (b) recombinant AAV virions,wherein the recombinant AAV virions comprise an AAV vector, the AAVvector comprising a selected gene operably linked to control elementscapable of directing the transcription and translation of the selectedgene when present in the subject, wherein the introducing is done invivo.

[0029] In another embodiment, the invention is directed to a method fordelivering a therapeutically effective amount of a protein systemicallyto a mammalian subject comprising:

[0030] (a) introducing a recombinant AAV virion into a muscle cell ortissue in vitro to produce a transduced muscle cell, wherein therecombinant AAV virion comprises an AAV vector, the AAV vectorcomprising a selected gene operably linked to control elements capableof directing the transcription and translation of the selected gene whenpresent in the subject; and

[0031] (b) administering to the subject a therapeutically effectiveamount of a composition comprising a pharmaceutically acceptableexcipient and the transduced muscle cells from step (a).

[0032] In other embodiments, the invention is directed to an AAV vectorcomprising a gene encoding either erythropoietin (EPO), or acidα-glucosidase (GAA), operably linked to control elements capable ofdirecting the in vivo transcription and translation of the gene, as wellas a recombinant AAV (rAAV) virion comprising the vector.

[0033] These and other embodiments of the subject invention will readilyoccur to those of ordinary skill in the art in view of the disclosureherein.

BRIEF DESCRIPTION OF THE FIGURES

[0034]FIG. 1 shows in situ histochemical detection of β-galactosidaseexpression in murine muscle cells following transduction with rAAV-LacZas described in Example 3, Part A. In the study, the tibialis anteriormuscle of adult Balb/c mice was injected with 8×10⁹ rAAV-LacZ. Animalswere sacrificed (a) 2, (b) 4, (c) 8, (d) 12, (e) 24, of (f) 32 weeksafter injection. The tibialis anterior was excised, and 10 mm sectionswere stained by X-gal for β-galactosidase histochemistry. The stainedtissue samples were photographed at 25×.

[0035]FIG. 2 shows a section of skeletal muscle two months afterinjection with rAAV-LacZ as described in Example 3, Part A. The tibialisanterior muscle was processed for in situ detection of β-galactosidaseexpression, and photographed with diffraction-interference contrastoptics at 400×.

[0036]FIG. 3 depicts β-galactosidase expression in Balb/c mice tibialisanterior muscle transduced in vivo with rAAV-LacZ as described inExample 3, Part B. Adult Balb/c mice were injected intramuscularly (IM)with various doses of rAAV-LacZ. At 2 and 8 weeks post injection, tissuewas harvested for analysis of beta-galactosidase (β-gal). β-galexpression was analyzed by measurement of relative light units (RLU)emitted from muscle homogenates, as detected by luminometer.

[0037]FIG. 4 shows the secretion of human erythropoietin (hEPO) fromtransduced myotubes and myoblasts, as described in Example 4. Myotubes(differentiated cells) or myoblasts (actively dividing cells) weretransduced with rAAV-hEPO at a ratio of approximately 10⁵ per targetcell. Levels of secreted hEPO were analyzed in supernatants at varioustime points. Baseline levels of hEPO prior to transduction were belowthe level of detection in both cell populations; the values at each timepoint represent replicate values +/−standard deviation.

[0038]FIG. 5 shows the secretion of human erythropoietin (hEPO) by C2C12myotubes transduced with rAAV-hEPO as described in Example 4. ConfluentC2C12 myoblasts were differentiated into myotubes and transduced with3×10⁸ (open bar), 3×10⁹ (cross-hatched bar), or 3×10¹⁰ (solid bar)rAAV-hEPO. Secretion of EPO was measured 3, 8, and 14 days aftertransduction. Control rAAV-LacZ myotubes secreted <2.5 mU/mL EPO. Thebar graph depicts mean production of EPO/well/24 hour as determined intriplicate cultures±the standard error of mean (SEM).

[0039]FIG. 6 shows the secretion of human erythropoietin (hEPO) byprimary human myotubes transduced with rAAV-hEPO as described in Example5. Confluent human myoblasts were differentiated into myotubes byculture for 14 days in reduced-serum media, then transduced with 3×10⁸(open bar), 3×10⁹ (cross-hatched bar), or 3×10¹⁰ (solid bar) rAAV-hEPO.Secretion of EPO was measured 3, 8 and 14 days after transduction.Control myotubes transduced with rAAV-LacZ secreted <2.5 mU/mL EPO. Thebar graph depicts mean production of EPO/well/24 hour as determined intriplicate cultures ±SEM.

[0040]FIG. 7 depicts the time course of EPO secretion in Balb/c miceafter IM injection with rAAV-hEPO. Adult Balb/c mice were injected IMwith 1×10¹⁰ (▾), 3×10¹⁰ (▴), 1×10¹¹ (▪), or 3×10¹¹ () purifiedrAAV-LacZ at day=0, and serum EPO levels measured at various time pointsafter injection. Reported values represent means (n=4)±SEM.

[0041]FIG. 8 shows high level expression of acid α-glucosidase (GAA) inhuman skeletal muscle transduced in vitro with rAAV-hGAA as described inExample 8, Part A. In the study, differentiated human myoblasts wereexposed to rAAV-hGAA virions at a MOI of 2×10⁵. Cells were collected atthe time points indicated, and GAA activity measured by enzymatic assay.Non-transduced control cells (open bar) and cells transduced withrAAV-LacZ (cross-hatched bar) showed no significant expression of GAA,while cells transduced with rAAV-hGAA (solid bar) showed high levels ofGAA activity. The bar graph represents mean GAA activity determined intriplicate cultures±SEM.

[0042]FIG. 9 shows expression of acid α-glucosidase in Balb/c micetibialis anterior muscle cells that were transduced in vivo withrAAV-hGAA, as described in Example 8, Part B. Adult Balb/c mice wereinjected intramuscularly (IM) with 4×10¹⁰ rAAV-hGAA (solid bar) or thesame dose of rAAV-LacZ (open bar). At various time points afterinjection, muscle tissue was harvested for analysis of GAA activity byenzymatic assay. The bar graph shows mean GAA activity determined infive animals (weeks 1 and 4), or in four animals (week 10)±SEM.

DETAILED DESCRIPTION OF THE INVENTION

[0043] The practice of the present invention will employ, unlessotherwise indicated, conventional methods of virology, microbiology,molecular biology and recombinant DNA techniques within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,Sambrook, et al. Molecular Cloning: A Laboratory Manual (CurrentEdition); DNA Cloning: A Practical Approach, vol. I & II (D. Glover,ed.); Oligonucleotide Synthesis (N. Gait, ed., Current Edition); NucleicAcid Hybridization (B. Hames & S. Higgins, eds., Current Edition);Transcription and Translation (B. Hames & S. Higgins, eds., CurrentEdition); CRC Handbook of Parvoviruses, vol. I & II (P. Tijssen, ed.);Fundamental Virology, 2nd Edition, vol. I & II (B. N. Fields and D. M.Knipe, eds.)

[0044] All publications, patents and patent applications cited herein,whether supra or infra, are hereby incorporated by reference in theirentirety.

[0045] As used in this specification and the appended claims, thesingular forms “a,” “an” and “the” include plural references unless thecontent clearly dictates otherwise.

[0046] A. Definitions

[0047] In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

[0048] The phrase “gene delivery” or “gene transfer” refers to methodsor systems for reliably inserting foreign DNA into target cells, such asinto muscle cells. Such methods can result in transient or long termexpression of genes. Gene transfer provides a unique approach for thetreatment of acquired and inherited diseases. A number of systems havebeen developed for gene transfer into mammalian cells. See, e.g., U.S.Pat. No. 5,399,346.

[0049] The term “therapeutic protein” refers to a protein which isdefective or missing from the subject in question, thus resulting in adisease state or disorder in the subject, or to a protein which confersa benefit to the subject in question, such as an antiviral,antibacterial or antitumor function. A therapeutic protein can also beone which modifies any one of a wide variety of biological functions,such as endocrine, immunological and metabolic functions. Representativetherapeutic proteins are discussed more fully below.

[0050] By “vector” is meant any genetic element, such as a plasmid,phage, transposon, cosmid, chromosome, virus, virion, etc., which iscapable of replication when associated with the proper control elementsand which can transfer gene sequences between cells. Thus, the termincludes cloning and expression vehicles, as well as viral vectors.

[0051] By “AAV vector” is meant a vector derived from anadeno-associated virus serotype, including without limitation, AAV-1,AAV-2, AAV-3, AAV-4, AAV-5, AAVX7, etc. AAV vectors can have one or moreof the AAV wild-type genes deleted in whole or part, preferably the repand/or cap genes (described below), but retain functional flanking ITRsequences (also described below). Functional ITR sequences are necessaryfor the rescue, replication and packaging of the AAV virion. Thus, anAAV vector is defined herein to include at least those sequencesrequired in cis for replication and packaging (e.g., functional ITRs) ofthe virus. The ITRs need not be the wild-type nucleotide sequences, andmay be altered, e.g., by the insertion, deletion or substitution ofnucleotides, so long as the sequences provide for functional rescue,replication and packaging.

[0052] By “recombinant virus” is meant a virus that has been geneticallyaltered, e.g., by the addition or insertion of a heterologous nucleicacid construct into the particle.

[0053] By “AAV virion” is meant a wild-type (wt) AAV virus particle(comprising a linear, single-stranded AAV nucleic acid genome associatedwith an AAV capsid protein coat). In this regard, single-stranded AAVnucleic acid molecules of either complementary sense, e.g., “sense” or“antisense” strands, can be packaged into any one AAV virion and bothstrands are equally infectious.

[0054] A “recombinant AAV virion,” or “rAAV virion” is defined herein asan infectious, replication-defective virus composed of an AAV proteinshell, encapsidating a DNA molecule of interest which is flanked on bothsides by AAV ITRs. An rAAV virion is produced in a suitable producercell which has had an AAV vector, AAV helper functions and accessoryfunctions introduced therein. In this manner, the producer cell isrendered capable of encoding AAV polypeptides that are required forpackaging the AAV vector (containing a recombinant nucleotide sequenceof interest) into recombinant virion particles for subsequent genedelivery.

[0055] The term “transfection” is used to refer to the uptake of foreignDNA by a mammalian cell. A cell has been “transfected” when exogenousDNA has been introduced inside the cell membrane. A number oftransfection techniques are known in the art. See, e.g., Graham et al.(1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, alaboratory manual, Cold Spring Harbor Laboratories, New York, Davis etal. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al.(1981) Gene 13:197. Such techniques can be used to introduce one or moreexogenous DNA moieties, such as a plasmid vector and other nucleic acidmolecules, into suitable cells. The term refers to both stable andtransient uptake of the genetic material.

[0056] The term “transduction” denotes the delivery of a DNA molecule toa recipient cell either in vivo or in vitro, via a replication-defectiveviral vector, such as via a recombinant AAV virion.

[0057] By “muscle cell” or “tissue” is meant a cell or group of cellsderived from muscle, including but not limited to cells and tissuederived from skeletal muscle; smooth muscle, e.g., from the digestivetract, urinary bladder and blood vessels; and cardiac muscle. The termcaptures muscle cells both in vitro and in vivo. Thus, for example, anisolated cardiomyocyte would constitute a “muscle cell” for purposes ofthe present invention, as would a muscle cell as it exists in muscletissue present in a subject in vivo. The term also encompasses bothdifferentiated and nondifferentiated muscle cells, such as myocytes,myotubes, myoblasts, cardipmyocytes and cardiomyoblasts.

[0058] The term “heterologous” as it relates to nucleic acid sequencessuch as gene sequences and control sequences, denotes sequences that arenot normally joined together, and/or are not normally associated with aparticular cell. Thus, a “heterologous” region of a nucleic acidconstruct or a vector is a segment of nucleic acid within or attached toanother nucleic acid molecule that is not found in association with theother molecule in nature. For example, a heterologous region of anucleic acid construct could include a coding sequence flanked bysequences not found in association with the coding sequence in nature.Another example of a heterologous coding sequence is a construct inwhich the coding sequence itself is not found in nature (e.g., syntheticsequences having codons different from the native gene). Similarly, acell transformed with a construct which is not normally present in thecell would be considered heterologous for purposes of this invention.Allelic variation or naturally occurring mutational events do not giverise to heterologous DNA, as used herein.

[0059] By “DNA” is meant a polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in double-stranded orsingle-stranded form, either relaxed and supercoiled. This term refersonly to the primary and secondary structure of the molecule, and doesnot limit it to any particular tertiary forms. Thus, this term includessingle- and double-stranded DNA found, inter alia, in linear DNAmolecules (e.g., restriction fragments), viruses, plasmids, andchromosomes. In discussing the structure of particular DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenontranscribed strand of DNA (i.e., the strand having the sequencehomologous to the mRNA). The term captures molecules that include thefour bases adenine, guanine, thymine, or cytosine, as well as moleculesthat include base analogues which are known in the art.

[0060] A “gene” or “coding sequence” or a sequence which “encodes” aparticular protein, is a nucleic acid molecule which is transcribed (inthe case of DNA) and translated (in the case of MRNA) into a polypeptidein vitro or in vivo when placed under the control of appropriateregulatory sequences. The boundaries of the gene are determined by astart codon at the 5′ (amino) terminus and a translation stop codon atthe 3′ (carboxy) terminus. A gene can include, but is not limited to,cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences fromprokaryotic or eukaryotic DNA, and even synthetic DNA sequences. Atranscription termination sequence will usually be located 3′ to thegene sequence.

[0061] The term “control elements” refers collectively to promoterregions, polyadenylation signals, transcription termination sequences,upstream regulatory domains, origins of replication, internal ribosomeentry sites (“IRES”), enhancers, and the like, which collectivelyprovide for the replication, transcription and translation of a codingsequence in a recipient cell. Not all of these control elements needalways be present so long as the selected coding sequence is capable ofbeing replicated, transcribed and translated in an appropriate hostcell.

[0062] The term “promoter region” is used herein in its ordinary senseto refer to a nucleotide region comprising a DNA regulatory sequence,wherein the regulatory sequence is derived from a gene which is capableof binding RNA polymerase and initiating transcription of a downstream(3′-direction) coding sequence.

[0063] “Operably linked” refers to an arrangement of elements whereinthe components so described are configured so as to perform their usualfunction. Thus, control elements operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol elements need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter sequence and the coding sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

[0064] For the purpose of describing the relative position of nucleotidesequences in a particular nucleic acid molecule throughout the instantapplication, such as when a particular nucleotide sequence is describedas being situated “upstream,” “downstream,” “3′,” or “5′” relative toanother sequence, it is to be understood that it is the position of thesequences in the “sense” or “coding” strand of a DNA molecule that isbeing referred to as is conventional in the art.

[0065] “Homology” refers-to the percent of identity between twopolynucleotide or two polypeptide moieties. The correspondence betweenthe sequence from one moiety to another can be determined by techniquesknown in the art. For example, homology can be determined by a directcomparison of the sequence information between two polypeptide moleculesby aligning the sequence information and using readily availablecomputer programs. Alternatively, homology can be determined byhybridization of polynucleotides under conditions which form stableduplexes between homologous regions, followed by digestion withsingle-stranded-specific nuclease(s), and size determination of thedigested fragments. Two DNA, or two polypeptide sequences are“substantially homologous” to each other when at least about 80%,preferably at least about 90%, and most preferably at least about 95% ofthe nucleotides or amino acids match over a defined length of themolecules, as determined using the methods above.

[0066] By “mammalian subject” is meant any member of the class Mammaliaincluding, without limitation, humans and nonhuman primates such aschimpanzees and other apes and monkey species; farm animals such ascattle, sheep, pigs, goats and horses; domestic mammals such as dogs andcats; laboratory animals including rodents such as mice, rats and guineapigs, and the like. The term does not denote a particular age or sex.Thus, adult and newborn subjects, as well as fetuses, whether male orfemale, are intended to be covered.

[0067] B. General Methods

[0068] The present invention provides for the successful transfer of aselected gene to a muscle cell using recombinant AAV virions. The methodallows for the direct, in vivo injection of recombinant AAV virions intomuscle tissue, e.g., by intramuscular injection, as well as for the invitro transduction of muscle cells which can subsequently be introducedinto a subject for treatment. The invention also provides for secretionof the produced protein in vivo, from transduced muscle cells, such thatsystemic delivery can be achieved.

[0069] Muscle provides a desirable target for gene therapy since musclecells are readily accessible and nondividing. However, the presentinvention also finds use with nondifferentiated muscle cells, such asmyoblasts, which can be transduced in vitro, and subsequently introducedinto a subject.

[0070] Since muscle has ready access to the circulatory system, aprotein produced and secreted by muscle cells and tissue in vivo willenter the bloodstream for systemic delivery. Furthermore, sincesustained, therapeutic levels of protein secretion from muscle isachieved in vivo using the present invention, repeated parenteraldelivery is avoided or reduced in frequency such that therapy can beaccomplished using only one or few injections. Thus, the presentinvention provides significant advantages over prior gene deliverymethods.

[0071] The recombinant AAV virions of the present invention, includingthe DNA of interest, can be produced using standard methodology, knownto those of skill in the art. The methods generally involve the steps of(1) introducing an AAV expression vector into a producer cell.; (2)introducing an AAV helper construct into the producer cell, where thehelper construct includes AAV coding regions capable of being expressedin the producer cell to complement AAV helper functions missing from theAAV vector; (3) introducing one or more helper viruses and/or accessoryfunction vectors into the producer cell, wherein the helper virus and/oraccessory function vectors provide accessory functions capable ofsupporting efficient recombinant AAV (“rAAV”) virion production in thecell; and (4) culturing the producer cell to produce rAAV virions. TheAAV expression vector, AAV helper construct and the helper virus oraccessory function vector(s) can be introduced into the producer cell,either simultaneously or serially, using standard transfectiontechniques.

[0072] 1. AAV Expression Vectors

[0073] AAV expression vectors are constructed using known techniques toat least provide as operatively linked components in the direction oftranscription, control elements including a transcriptional initiationregion, the DNA of interest and a transcriptional termination region.The control elements are selected to be functional in a mammalian musclecell. The resulting construct which contains the operatively linkedcomponents is bounded (5′ and 3′) with functional AAV ITR sequences.

[0074] The nucleotide sequences of AAV ITR regions are known. See, e.g.,Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Berns, K. I.“Parvoviridae and their Replication” in Fundamental Virology, 2ndEdition, (B. N. Fields and D. M. Knipe, eds.) for the AAV-2 sequence.AAV ITRs used in the vectors of the invention need not have a wild-typenucleotide sequence, and may be altered, e.g., by the insertion,deletion or substitution of nucleotides. Additionally, AAV ITRs may bederived from any of several AAV serotypes, including without limitation,AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAVX7, etc. Furthermore, 5′ and 3′ITRs which flank a selected nucleotide sequence in an AAV expressionvector need not necessarily be identical or derived from the same AAVserotype or isolate, so long as they function as intended, i.e., toallow for packaging of virions.

[0075] Suitable DNA molecules for use in AAV vectors will be less thanabout 5 kilobases (kb) in size and will include, for example, a genethat encodes a protein that is defective or missing from a recipientsubject or a gene that encodes a protein having a desired biological ortherapeutic effect (e.g., an antibacterial, antiviral or antitumorfunction).

[0076] Suitable DNA molecules include, but are not limited to, thoseencoding for proteins used for the treatment of endocrine, metabolic,hematologic, cardiovascular, neurologic, musculoskeletal, urologic,pulmonary and immune disorders, including such disorders as inflammatorydiseases, autoimmune, chronic and infectious diseases, such as AIDS,cancer, hypercholestemia, insulin disorders such as diabetes, growthdisorders, various blood disorders including various anemias,thalassemias and hemophilia; genetic defects such as cystic fibrosis,Gaucher's Disease, Hurler's Disease, adenosine deaminase (ADA)deficiency, emphysema, or the like.

[0077] To exemplify the invention, the gene encoding erythropoietin(EPO) can be used. EPO is a glycoprotein hormone produced in fetal liverand adult kidney which acts on progenitor cells in the bone marrow andother hematopoietic tissue to stimulate the formation of red bloodcells. Genes encoding human and other mammalian EPO have been cloned,sequenced and expressed, and show a high degree of sequence homology inthe coding region across species. Wen et al. (1993) Blood 82:1507-1516.The sequence of the gene encoding native human EPO, as well as methodsof obtaining the same, are described in, e.g., U.S. Pat. Nos. 4,954,437and 4,703,008, incorporated herein by reference in their entirety, aswell as in Jacobs et al. (1985) Nature 313:806-810; Lin et al. (1985)Proc. Natl. Acad. Sci. USA 82:7580; International Publication Number WO85/02610; and European Patent Publication Number 232,034-B1. Inaddition, the sequences of the genes encoding native feline, canine andporcine EPO are known and readily available (GenBank Accession Nos.:L10606; L13027; and L10607, respectively), and the sequence of the geneencoding monkey (Macaca mulatta) is also known and available (GenBankAccession No.: L10609). The term “EPO” as used herein refers to thenative, full-length secreted form of EPO, as well as to analogs orderivatives thereof comprising single or multiple amino acidsubstitutions, deletions or additions which retain EPO function oractivity. In this regard, a number of small peptides have beenidentified which bind to and activate the receptor for EPO. Wrighton etal. (1996) Science 273:458-463; Livnah et al. (1996) Science273:464-471. The recombinant AAV virions described herein which includea gene encoding EPO, or encoding an analog or derivative thereof havingthe same function, are particularly useful in the treatment of blooddisorders characterized by defective red blood cell formation, such asin the treatment of anemia. Increased red blood cell production due tothe production of EPO can be readily determined by an appropriateindicator, such as by comparing hematocrit measurements pre- andpost-treatment, measuring increases in red blood cell count, hemoglobinconcentration, or in reticulocyte counts. As described above, the EPOgene is flanked by AAV ITRs.

[0078] Alternatively, a nucleotide sequence encoding the lysosomalenzyme acid α-glucosidase (GAA) can be used. GAA functions to cleaveα-1,4 and α-1,6 linkages of lysosomal glycogen to releasemonosaccharides. The sequence of the gene encoding human GAA, as well asmethods of obtaining the same, have been previously described (GenBankAccession Numbers: M34424 and Y00839; Martiniuk et al. (1990) DNA CellBiol. 9:85-94; Martiniuk et al. (1986) Proc. Natl. Acad. Sci. USA83:9641-9644; Hoefsloot et al. (1988) Eur. Mol. Biol. Organ.7:1697-1704). Thus, the recombinant AAV virions described herein caninclude a nucleotide sequence encoding GAA, or encoding an analog orderivative thereof having GAA activity.

[0079] The selected nucleotide sequence, such as EPO or another gene ofinterest, is operably linked to control elements that direct thetranscription or expression thereof in the subject in vivo. Such controlelements can comprise control sequences normally associated with theselected gene. Alternatively, heterologous control sequences can beemployed. Useful heterologous control sequences generally include thosederived from sequences encoding mammalian or viral genes. Examplesinclude, but are not limited to, the SV40 early promoter; mouse mammarytumor virus LTR promoter; adenovirus major late promoter (Ad MLP);herpes simplex virus (HSV) promoters; a cytomegalovirus (CMV) promotersuch as the CMV immediate early promoter region (CMVIE); a rous sarcomavirus (RSV) promoter; synthetic promoters; hybrid promoters; and thelike. In addition, sequences derived from nonviral genes, such as themurine metallothionein gene, will also find use herein. Such promotersequences are commercially available from, e.g., Stratagene (San Diego,Calif.).

[0080] For purposes of the present invention, control elements, such asmuscle-specific and inducible promoters, enhancers and the like, will beof particular use. Such control elements include, but are not limitedto, those derived from the actin and myosin gene families, such as fromthe myoD gene family (Weintraub et al. (1991) Science 251:761-766); themyocyte-specific enhancer binding factor MEF-2 (Cserjesi and Olson(1991) Mol. Cell Biol. 11:4854-4862); control elements derived from thehuman skeletal actin gene (Muscat et al. (1987) Mol. Cell Biol.7:4089-4099) and the cardiac actin gene; muscle creatine kinase sequenceelements (Johnson et al. (1989) Mol. Cell Biol. 9:3393-3399) and themurine creatine kinase enhancer (mCK) element; control elements derivedfrom the skeletal fast-twitch troponin C gene, the slow-twitch cardiactroponin C gene and the slow-twitch troponin I gene; hypoxia-induciblenuclear factors (Semenza et al. (1991) Proc. Natl. Acad. Sci. USA88:5680-5684; Semenza et al. J. Biol. Chem. 269:23757-23763);steroid-inducible elements and promoters, such as the glucocorticoidresponse element (GRE) (Mader and White (1993) Proc. Natl. Acad. Sci.USA 90:5603-5607); the fusion consensus element for RU486 induction;elements that provide for tetracycline regulated gene expression (Dhawanet al. (1995) Somat. Cell. Mol. Genet. 21:233-240; Shockett et al.(1995) Proc. Natl. Acad. Sci. USA 92:6522-6526; and inducible, synthetichumanized promoters (Rivera et al. (1996) Nature Med. 2:1028-1032).

[0081] These and other regulatory elements can be tested for potentialin vivo efficacy using the in vitro myoblast model, which mimicsquiescent in vivo muscle physiology, described in the examples below.

[0082] The AAV expression vector which harbors the DNA molecule ofinterest bounded by AAV ITRs, can be constructed by directly insertingthe selected sequence(s) into an AAV genome which has had the major AAVopen reading frames (“ORFs”) excised therefrom. Other portions of theAAV genome can also be deleted, so long as a sufficient portion of theITRs remain to allow for replication and packaging functions. Suchconstructs can be designed using techniques well known in the art. See,e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International PublicationNos. WO 92/01070 (published Jan. 23, 1992) and WO 93/03769 (publishedMar. 4, 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539;Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shellingand Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp.Med. 179:1867-1875.

[0083] Alternatively, AAV ITRs can be excised from the viral genome orfrom an AAV vector containing the same and fused 5′ and 3′ of a selectednucleic acid construct that is present in another vector using standardligation techniques, such as those described in Sambrook et al., supra.For example, ligations can be accomplished in 20 mM Tris-Cl pH 7.5, 10mM MgCl₂, 10 mM DTT, 33 μg/ml BSA, 10 mM-50 mM NaCl, and either 40 μMATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0° C. (for “sticky end”ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C.(for “blunt end” ligation). Intermolecular “sticky end” ligations areusually performed at 30-100 μg/ml total DNA concentrations (5-100 nMtotal end concentration). AAV vectors which contain ITRs have beendescribed in, e.g., U.S. Pat. No. 5,139,941. In particular, several AAVvectors are described therein which are available from the American TypeCulture Collection (“ATCC”) under Accession Numbers 53222, 53223, 53224,53225 and 53226.

[0084] Additionally, chimeric genes can be produced synthetically toinclude AAV ITR sequences arranged 5′ and 3′ of one or more selectednucleic acid sequences. Preferred codons for expression of the chimericgene sequence in mammalian muscle cells can be used. The completechimeric sequence is assembled from overlapping oligonucleotidesprepared by standard methods. See, e.g., Edge, Nature (1981) 292:756;Nambair et al. Science (1984) 223:1299; Jay et al. J. Biol. Chem. (1984)259:6311.

[0085] In order to produce rAAV virions, an AAV expression vector isintroduced into a suitable producer cell using known techniques, such asby transfection. A number of transfection techniques are generally knownin the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrooket al. (1989) Molecular Cloning, a laboratory manual, Cold Spring HarborLaboratories, New York, Davis et al. (1986) Basic Methods in MolecularBiology, Elsevier, and Chu et al. (1981) Gene 13:197. Particularlysuitable transfection methods include calcium phosphate co-precipitation(Graham et al. (1973) Virol. 52:456-467), direct micro-injection intocultured cells (Capecchi, M. R. (1980) Cell 22:479-488), electroporation(Shigekawa et al. (1988) BioTechniques 6:742-751), liposome mediatedgene transfer (Mannino et al. (1988) BioTechniques 6:682-690),lipid-mediated transduction (Felgner et al. (1987) Proc. Natl. Acad.Sci. USA 84:7413-7417), and nucleic acid delivery using high-velocitymicroprojectiles (Klein et al. (1987) Nature 327:70-73).

[0086] For the purposes of the invention, suitable producer cells forproducing rAAV virions include microorganisms, yeast cells, insectcells, and mammalian cells, that can be, or have been, used asrecipients of a heterologous DNA molecule. The term includes the progenyof the original cell which has been transfected. Thus, a “producer cell”as used herein generally refers to a cell which has been transfectedwith an exogenous DNA sequence. Cells from the stable human cell line,293 (readily available through, e.g., the American Type CultureCollection under Accession Number ATCC CRL1573) are preferred in thepractice of the present invention. Particularly, the human cell line 293is a human embryonic kidney cell line that has been transformed withadenovirus type-5 DNA fragments (Graham et al. (1977) J. Gen. Virol.36:59), and expresses the adenoviral Ela and Elb genes (Aiello et al.(1979) Virology 94:460). The 293 cell line is readily transfected, andprovides a particularly convenient platform in which to produce rAAVvirions.

[0087] 2. AAV Helper Functions

[0088] Producer cells containing the above-described AAV expressionvectors must be rendered capable of providing AAV helper functions inorder to replicate and encapsidate the nucleotide sequences flanked bythe AAV ITRs to produce rAAV virions. AAV helper functions are generallyAAV-derived coding sequences which can be expressed to provide AAV geneproducts that, in turn, function in trans for productive AAVreplication. AAV helper functions are used herein to complementnecessary AAV functions that are missing from the AAV expressionvectors. Thus, AAV helper functions include one, or both of the majorAAV ORFs, namely the rep and cap coding regions, or functionalhomologues thereof.

[0089] By “AAV rep coding region” is meant the art-recognized region ofthe AAV genome which encodes the replication proteins Rep 78, Rep 68,Rep 52 and Rep 40. These Rep expression products have been shown topossess many functions, including recognition, binding and nicking ofthe AAV origin of DNA replication, DNA helicase activity and modulationof transcription from AAV (or other heterologous) promoters. The Repexpression products are collectively required for replicating the AAVgenome. For a description of the AAV rep coding region, see, e.g.,Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801.Suitable homologues of the AAV rep coding region include the humanherpesvirus 6 (HHV-6) rep gene which is also known to mediate AAV-2 DNAreplication (Thomson et al. (1994) Virology 204:304-311).

[0090] By “AAV cap coding region” is meant the art-recognized region ofthe AAV genome which encodes the capsid proteins VP1, VP2, and VP3, orfunctional homologues thereof. These cap expression products are thecapsid proteins which are collectively required for packaging the viralgenome. For a description of the AAV cap coding region, see, e.g.,Muzyczka, N. and Kotin, R. M. (supra).

[0091] AAV helper functions are introduced into the producer cell bytransfecting the cell with an AAV helper construct either prior to, orconcurrently with, the transfection of the AAV expression vector. AAVhelper constructs are thus used to provide at least transient expressionof AAV rep and/or cap genes to complement missing AAV functions that arenecessary for productive AAV infection. AAV helper constructs lack AAVITRs and can neither replicate nor package themselves. These constructscan be in the form of a plasmid, phage, transposon, cosmid, virus, orvirion. A number of AAV helper constructs have been described, such asthe commonly used plasmids pAAV/Ad and pIM29+45 which encode both Repand Cap expression products. See, e.g., Samulski et al. (1989) J. Virol.63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945. A numberof other vectors have been described which encode Rep and/or Capexpression products. See, e.g., U.S. Pat. No. 5,139,941.

[0092] Both AAV expression vectors and AAV helper constructs can beconstructed to contain one or more optional selectable markers. Suitablemarkers include genes which confer antibiotic resistance or sensitivityto, impart color to, or change the antigenic characteristics of thosecells which have been transfected with a nucleic acid constructcontaining the selectable marker when the cells are grown in anappropriate selective medium. Several selectable marker genes that areuseful in the practice of the invention include the hygromycin Bresistance gene (encoding Aminoglycoside phosphotranferase (APH)) thatallows selection in mammalian cells by conferring resistance to G418(available from Sigma, St. Louis, Mo.). Other suitable markers are knownto those of skill in the art.

[0093] 3. Accessory Functions

[0094] The producer cell must also be rendered capable of providing nonAAV derived functions, or “accessory functions,” in order to producerAAV virions. Accessory functions are non AAV derived viral and/orcellular functions upon which AAV is dependent for its replication.Thus, accessory functions include at least those non AAV proteins andRNAs that are required in AAV replication, including those involved inactivation of AAV gene transcription, stage specific AAV MRNA splicing,AAV DNA replication, synthesis of Cap expression products and AAV capsidassembly. Viral-based accessory functions can be derived from any of theknown helper viruses.

[0095] Particularly, accessory functions can be introduced into and thenexpressed in producer cells using methods known to those of skill in theart. Commonly, accessory functions are provided by infection of theproducer cells with an unrelated helper virus. A number of suitablehelper viruses are known, including adenoviruses; herpesviruses such asherpes simplex virus types 1 and 2; and vaccinia viruses. Nonviralaccessory functions will also find use herein, such as those provided bycell synchronization using any of various known agents. See, e.g.,Buller et al. (1981) J. Virol. 40:241-247; McPherson et al. (1985)Virology 147:217-222; Schlehofer et al. (1986) Virology 152:110-117.

[0096] Alternatively, accessory functions can be provided using anaccessory function vector. Accessory function vectors include nucleotidesequences that provide one or more accessory functions. An accessoryfunction vector is capable of being introduced into a suitable producercell in order to support efficient AAV virion production in the cell.Accessory function vectors can be in the form of a plasmid, phage,transposon or cosmid. Accessory vectors can also be in the form of oneor more linearized DNA or RNA fragments which, when associated with theappropriate control elements and enzymes, can be transcribed orexpressed in a producer cell to provide accessory functions.

[0097] Nucleic acid sequences providing the accessory functions can beobtained from natural sources, such as from the genome of an adenovirusparticle, or constructed using recombinant or synthetic methods known inthe art. In this regard, adenovirus-derived accessory functions havebeen widely studied, and a number of adenovirus genes involved inaccessory functions have been identified and partially characterized.See, e.g., Carter, B. J. (1990) “Adeno-Associated Virus HelperFunctions,” in CRC Handbook of Parvoviruses, vol. I (P. Tijssen, ed.),and Muzyczka, N. (1992) Curr. Topics. Microbiol. and Immun. 158:97-129.Specifically, early adenoviral gene regions Ela, E2a, E4, VAI RNA and,possibly, E1b are thought to participate in the accessory process. Janiket al. (1981) Proc. Natl. Acad. Sci. USA 78:1925-1929.Herpesvirus-derived accessory functions have been described. See, e.g.,Young et-al. (1979) Prog. Med. Virol. 25:113. Vaccinia virus-derivedaccessory functions have also been described. See, e.g., Carter, B.J.(1990), supra., Schlehofer et al. (1986) Virology 152:110-117.

[0098] As a consequence of the infection of the producer cell with ahelper virus, or transfection of the producer cell with an accessoryfunction vector, accessory functions are expressed which transactivatethe AAV helper construct to produce AAV Rep and/or Cap proteins. The Repexpression products excise the recombinant DNA (including the DNA ofinterest) from the AAV expression vector. The Rep proteins also serve toduplicate the AAV genome. The expressed Cap proteins assemble intocapsids, and the recombinant AAV genome is packaged into the capsids.Thus, productive AAV replication ensues, and the DNA is packaged intorAAV virions.

[0099] Following recombinant AAV replication, rAAV virions can bepurified from the producer cell using a variety of conventionalpurification methods, such as CsCl gradients. Further, if infection isemployed to express the accessory functions, residual helper virus canbe inactivated, using known methods. For example, adenovirus can beinactivated by heating to temperatures of approximately 60° C. for,e.g., 20 minutes or more. This treatment effectively inactivates onlythe helper virus since AAV is extremely heat stable while the helperadenovirus is heat labile.

[0100] The resulting rAAV virions are then ready for use for DNAdelivery, such as in gene therapy applications, for the production oftransgenic animals, in vaccination, and particularly for the delivery ofgenes to a variety of muscle cell types.

[0101] 4. In Vitro and In Vivo Delivery of rAAV Virions

[0102] Generally, rAAV virions are introduced into a muscle cell usingeither in vivo or in vitro transduction techniques. If transduced invitro, the desired recipient muscle cell will be removed from thesubject, transduced with rAAV virions and reintroduced into the subject.Alternatively, syngeneic or xenogeneic muscle cells can be used wherethose cells will not generate an inappropriate immune response in thesubject.

[0103] Suitable methods for the delivery and introduction of transducedcells into a subject have been described. For example, cells can betransduced in vitro by combining recombinant AAV virions with musclecells e.g., in appropriate media, and screening for those cellsharboring the DNA of interest using conventional techniques such asSouthern blots and/or PCR, or by using selectable markers. Transducedcells can then be formulated into pharmaceutical compositions, describedmore fully below, and the composition introduced into the subject byvarious techniques, such as by intramuscular, intravenous, subcutaneousand intraperitoneal injection, or by injection into smooth and cardiacmuscle, using e.g., a catheter.

[0104] For in vivo delivery, the rAAV virions will be formulated intopharmaceutical compositions and will generally be administeredparenterally, e.g., by intramuscular injection directly into skeletal orcardiac muscle.

[0105] Pharmaceutical compositions will comprise sufficient geneticmaterial to produce a therapeutically effective amount of the protein ofinterest, i.e., an amount sufficient to reduce or ameliorate symptoms ofthe disease state in question or an amount sufficient to confer thedesired benefit. The pharmaceutical compositions will also contain apharmaceutically acceptable excipient. Such excipients include anypharmaceutical agent that does not itself induce the production ofantibodies harmful to the individual receiving the composition, andwhich may be administered without undue toxicity. Pharmaceuticallyacceptable excipients include, but are not limited to, liquids such aswater, saline, glycerol and ethanol. Pharmaceutically acceptable saltscan be included therein, for example, mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like; andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. Additionally, auxiliary substances, such aswetting or emulsifying agents, pH buffering substances, and the like,may be present in such vehicles. A thorough discussion ofpharmaceutically acceptable excipients is available in REMINGTON'SPHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991).

[0106] Appropriate doses will depend on the mammal being treated (e.g.,human or nonhuman primate or other mammal), age and general condition ofthe subject to be treated, the severity of the condition being treated,the particular therapeutic protein in question, its mode ofadministration, among other factors. An appropriate effective amount canbe readily determined by one of skill in the art.

[0107] Thus, a “therapeutically effective amount” will fall in arelatively broad range that can be determined through clinical trials.For example, for in vivo injection, i.e., injection directly to skeletalor cardiac muscle, a therapeutically effective dose will be on the orderof from about 10⁶ to 10¹⁵ of the rAAV virions, more preferably 10⁸ to10¹⁴ rAAV virions. For in vitro transduction, an effective amount ofrAAV virions to be delivered to muscle cells will be on the order of 10⁸to 10¹³ of the rAAV virions. The amount of transduced cells in thepharmaceutical compositions will be from about 10⁴ to 10¹⁰ muscle cells,more preferably 10⁵ to 10⁸ muscle cells. When the transduced cells areintroduced to vascular smooth muscle, a lower dose may be appropriate.Other effective dosages can be readily established by one of ordinaryskill in the art through routine trials establishing dose responsecurves.

[0108] Dosage treatment may be a single dose schedule or a multiple doseschedule. Moreover, the subject may be administered as many doses asappropriate. One of skill in the art can readily determine anappropriate number of doses.

[0109] C. Experimental

[0110] Below are examples of specific embodiments for carrying out thepresent invention. The examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

[0111] Efforts have been made to ensure accuracy with respect to numbersused (e.g., amounts, temperatures, etc.), but some experimental errorand deviation should, of course, be allowed for.

Materials and Methods

[0112] Vector Constructs

[0113] A. Construction of p1909adhlacZ.

[0114] Plasmid p1909adhlacZ was used as the helper construct in thefollowing examples and was constructed from plasmid pwadhlacz. PlasmidpWadhlacZ was constructed by partially digesting plasmid pUC119(GeneBank Reference Name: U07649, GeneBank Accession Number: U07649)with AflIII and BspHI, blunt-end modifying with the klenow enzyme, andthen ligating to form a circular 1732 bp plasmid containing thebacterial origin and the amp gene only (the polylinker and Fl origin wasremoved). The blunted and ligated AflIII and BspHI junction forms aunique NspI site. The 1732 bp plasmid was cut with NspI, blunt-endmodified with T4 polymerase, and a 20 bp HinDIII-HinCII fragment(blunt-end modified with the klenow enzyme) obtained from the pUC119polylinker was ligated into the blunted NspI site of the plasmid. TheHinDIII site from the blunted polylinker was regenerated, and thenpositioned adjacent to the bacterial origin of replication. Theresulting plasmid was then cut at the unique PstI/See8387I site, and anSse8387I-PvuII-Sse8387I oligonucleotide, having the sequence:5′-GGCAGCTGCCTGCA-3′ (SEQ ID NO.:______), was ligated therein. Theremaining unique BspHI site was cut, blunt-end modified with klenowenzyme, and an AscI linker oligonucleotide, having the sequence:5′-GAAGGCGCGCCTTC-3′ (SEQ ID NO.:______) was ligated therein,eliminating the BspHI site. The resulting plasmid was called pWee.

[0115] In order to create the pWadhlacZ construct, a CMVlacZ expressioncassette (comprising a nucleotide sequence flanked 5′ and 3′ by AAVITRs, containing the following elements: a CMV promoter, the hGH 1stintron, an adhlacz fragment and an SV40 early polyadenylation site) wasinserted into the unique PvuII site of pWee using multiple steps suchthat the CMV promoter was arranged proximal to the bacterial amp gene ofpWee.

[0116] More particularly, a CMVlacZ expression cassette was derived fromthe plasmid psub201CMV, which was constructed as follows. Anoligonucleotide encoding the restriction enzyme sites:

[0117] NotI-MluI-SnaBI-AgeI-BstBI-BssHII-NcoI-HpaI-BspEI-PmlI-RsrII-NotI

[0118] and having the following nucleotide sequence:

[0119] 5′-GCGGCCGCACGCGTACGTACCGGTTCGAAGCGCGCACGGCCGACCATGGTTAACTCCGGACACGTGCGGACCGCGGCCGC-3′ (SEQ ID No.:______) was synthesized andcloned into the blunt-end modified KasI-EarI site (partial) of pUC119 toprovide a 2757 bp vector fragment. A 653 bp SpeI-SacII fragmentcontaining a nucleotide sequence encoding a CMV immediate early promoterwas cloned into the SnaBI site of the 2757 bp vector fragment. Further,a 269 bp PCR-produced BstBI-BstBI fragment containing a nucleotidesequence encoding the hGH 1st intron which was derived using thefollowing primers:

[0120] 5-AAAATTCGAACCTGGGGAGAAACCAGAG-3′ (SEQ ID NO.:______) and

[0121] 3′-aaaattcgaacaggtaagcgcccctTTG-5′ (SEQ ID NO.:______), wascloned into the BstBI site of the 2757 bp vector fragment, and a 135 bpHpaI-BamHI (blunt-end modified) fragment containing the SV40 earlypolyadenylation site from the pCMV-β plasmid (CLONETECH) was cloned intothe HpaI site of the subject vector fragment. The resulting constructwas then cut with NotI to provide a first CMV expression cassette.

[0122] Plasmid pW1909adhlacZ was constructed as follows. A 4723 bpSpeI-EcoRV fragment containing the AAV rep and cap encoding region wasobtained from the plasmid pGN1909 (ATCC Accession Number 69871). ThepGN1909 plasmid is a high efficiency AAV helper plasmid having AAV repand cap genes with an AAV p5 promoter region that is arranged in theconstruct to be downstream from its normal position (in the wild typeAAV genome) relative to the rep coding region. The 4723 bp fragment wasblunt-end modified, and AscI linkers were ligated to the blunted ends.The resultant fragment was then ligated into the unique AscI site ofpWadhlacZ and oriented such that the AAV coding sequences were arrangedproximal to the bacterial origin of replication in the construct.

[0123] Plasmid pW1909adhlacZ includes the bacterial beta-galactosidase(#-gal) gene under the transcriptional control of the cytomegalovirusimmediate early promoter (CMVIE).

[0124] B. Construction of pW1909EPO.

[0125] Plasmid pW1909adhlacZ was modified to express humanerythropoietin (EPO) by replacing the adhlacz gene with a 718 base pairPpuMI-NcoI fragment of human EPO cDNA (Wen et al. (1993) Blood5:1507-1516) and by cloning a 2181 bp ClaI-EcoRI lacZ spacer fragment(noncoding) into the PmlI site of the vector.

[0126] C. Construction of pAAV-GAA.

[0127] A plasmid containing the human lysosomal enzyme acidα-glucosidase (GAA) coding region was constructed as follows. A 3.2 kBcDNA clone containing the coding sequence for human GAA beginning 207bps downstream from the initiation codon (GenBank Accession Numbers:M34424 and Y00839; Martiniuk et al. (1990) DNA Cell Biol. 9:85-94) wascloned into the EcoRI site of Bluescript KS (Stratagene). Additional 5′sequence was generated using polymerase chain reaction (PCR) withreverse-transcribed poly-A mRNA (obtained from normal human fibroblasts)as the template. The 5′ primer was constructed with a KpnI restrictionsite and bps-3 to 12 of the published sequence (Martiniuk et al. (1986)Proc. Natl. Acad. Sci. USA 83:9641-9644; Hoefsloot et al. (1988) Eur.Mol. Biol. Organ. 7-:1697-1704). The 3′ primer was synthesized frombasepairs 1001 to 1018. Using KpnI and the unique internal StuI site atposition 776, the PCR product was ligated to the partial cDNA to formthe full-length GAA-encoding plasmid. The full length cDNA was truncatedat a unique SphI restriction site, and cloned into the expression vectorp1.1c to result in the PAAV-GAA construct having the GAA coding regionunder transcriptional control of the CMV-IE promoter.

[0128] The p1.1c expression vector was constructed as follows. pUC119was partially digested with KasI and EarI, and a 2713 bp vector fragmentcontaining the ampicillin resistance gene, the coli 1 origin ofreplication and the M13 origin of replication, was isolated, blunt endmodified, and ligated to a synthetic DNA polylinker encoding therestriction enzyme sitesNotI-MluI-SnaBI-AgeI-SfuI-BssHII-EagI-NcoI-PmeI-BspEI-PmlI-RsrII-NotI,and having the following nucleotide sequence:

[0129] 5′-GCGGCCGCACGCGTTGTTAACAACCGGTTCGAAGCGCGCAGCGGCCGACCATGGGTTTAAACTCCGGACCACGTGCGGACCGAGCGGCCGC-3′ (SEQ IDNO.:______). The ligation was conducted such that the MluI end of thepolylinker was ligated to the KasI side of the plasmid. A 653 bpSpeI-SacII fragment encoding the CMV immediate-early promoter, a 269 bpPCR-produced SfuI-SfuI produced fragment encoding the hGH 1st intron(derived using the following primers:

[0130] 5′-AAAATTCGAACAGGTAAGCGCCCCTTTG-3′ (SEQ ID NO.:______) and3′-AAAATTCGAACCTGGGGAGAAACCAGAG-5′ (SEQ ID NO.:______)), a 183 bpBssHII-BssHII polylinker fragment from pbluescript II SK-, and a 135 bpHpaI-BamHI (blunted) fragment containing the SV40 early polyadenylationsite from pCMV-β (Stratagene), were cloned into the SnaBI, SfuI, BssHII,and PmeI sites, respectively, of the aforementioned plasmid. Theorientation of the polylinker relative to the intron and polyadenylationsite was intron-polylinker (5′SacI-3′KpnI)-polyadenylation site. Thepolylinker was further modified by removing the 88 bp SacI-XhoIpolylinker fragment and replacing it with the following synthetic SacIto XhoI fragment encoding the restriction enzyme sitesSacI-ClaI-EcoRI-SmaI-BamHI-XbaI-SalI-PstI-BstXI-EcoRV-BstXI-olmeganuclease-HinDIII-XhoI,having the following nucleotide sequence:

[0131] 5′-GAGCTCAATCGATTGAATTCCCCGGGGATCCTCTAGAGTCGACCTGCAGCCACTGTGTTGGATATCCAACACACTGGTAGGGATAACAGGGTAATCTCGAG-3′ (SEQ ID NO.:______).

[0132] Viruses and Cell Lines

[0133] Adenovirus type 2 (Ad2), available from the American Type CultureCollection, ATCC, Catalogue Number VR846, was used as helper virus toencapsidate vectors.

[0134] The human 293 cell line (Graham et al. (1977) J. Gen. Virol.36:59-72, available from the ATCC under Accession no. CRL1573), whichhas adenovirus E1a and E1b genes stably integrated in its genome, wascultured in complete Dulbecco's modified Eagle's media (DMEM;Bio-Whittaker, Walkersville, Md.) containing 4.5 g/L glucose, 10%heat-inactivated fetal bovine serum (FBS; Hyclone, Logan, Utah), 2 mMglutamine, and 50 units/mL penicillin and 50 μg/mL streptomycin.

[0135] The C2C12 murine myoblast cell line, available from the ATCC,Catalogue Number CRL1772, was cultured in DMEM with 20% fetal calf serum(FCS), 1 chick embryo extract and 5 μg/mL gentamicin.

[0136] Fetal human skeletal myoblasts (Clonetics) were cultured in HamsF-12 human growth medium, containing 20% FCS and 5 μg/mL gentamicin.

[0137] The above cell lines were incubated at 37° C. in 5% CO₂, and wereroutinely tested and found free of mycoplasma contamination.

[0138] Production of Recombinant AAV Virions

[0139] Recombinant AAV virions were produced in human 293 cells asfollows. Subconfluent 293 cells were cotransfected by standard calciumphosphate precipitation (Wigler et al. (1979) Proc. Natl. Acad. Sci USA76:1373-1376) with one of the AAV vector/helper plasmid constructs,pW1909adhLacZ or pW1909EPO; or with pAAV-GAA and the pW1909 helperplasmid. After 6 hours, the transfected cells were infected with Ad2 infresh medium at a multiplicity of infection (MOI) of 2, and incubated at37° C. in 5% CO₂ for 70 hours prior to harvest. Pelleted cells werelysed in Tris buffer (10 mM Tris, 150 mM NaCl, pH 8.0) by three cyclesof freeze-thaw. The lysate was clarified of cell debris bycentrifugation at 12,000×g, and the crude-cell lysate was layered onto acesium chloride cushion for isopyknic gradient centrifugation.Recombinant AAV virions (rAAV-LacZ, rAAV-hEPO, or rAAV-hGAA virions)were extracted from the resulting gradient by isolating the fractionswith an average density of approximately 1.38 g/mL, resuspended in Hepesbuffered saline (HBS) containing 50 mM Hepes (pH 7.4) and 150 mM NaCl.The preparations were then heated at 56° C. for approximately 1 hour toinactivate Ad2.

[0140] Assay of rAAV by Dot-Blot Hybridization

[0141] Recombinant AAV virions were DNase I digested, proteinase Ktreated, phenol-chloroform extracted, and DNA precipitated with sodiumacetate-glycogen (final concentrations=0.3 M sodium acetate and 160μg/mL, respectively). DNA samples were denatured (200 μL of 2× alkalinesolution (0.8 M NaOH, 20 mM EDTA) added to DNA sample) for 10 minutes,then added to appropriate wells in a dot-blot apparatus, and blottedonto wet Zeta Probe membrane (BioRad), by applying suction until wellswere empty. Then, 400 μL of 1× alkaline solution was added; after 5minutes, wells were emptied by suction. The membrane was rinsed in 2×SSC(Sambrook et al., supra) for 1 min, drained, air dried on filter paper,then baked in vacuum at 80° C. for 30 min. The membrane was thenprehybridized for 30 min at 65° C. with 10 mL hybridization buffer (7%SDS, 0.25 M Sodium Phosphate, pH 7.2, 1 mM EDTA). Buffer was replacedwith 10 mL fresh solution, freshly boiled probe added, and hybridizedovernight at 65° C. The membrane was washed twice with 25 mL of wash-1buffer (5% SDS, 40 mM sodium phosphate, pH 7.2, 1 mM EDTA) for 20 min at65° C. and twice with wash-2 buffer (1% SDS, 40 mM sodium phosphate, pH7.2, 1 mM EDTA). The membrane was wrapped in plastic film, exposed toradiographic film, and appropriate dots excised from the membrane todetermine radioactivity by scintillation counting, and quantitated bycomparison with standards. Titers of rAAV virion were routinely in therange of approximately 10¹³ genomes/mL.

[0142] Assay for Contaminating Helper Adenovirus

[0143] Contaminating infectious adenovirus was assayed as follows.Samples from the purified rAAV virion stocks were added to 50% confluent293 cells (cultured in 12 well dishes at 1×10⁵ cells/well), and thecultures were passaged for 30 days (e.g., the cultures were split 1 to5, every 3 days) or until the culture exhibited 100% cytopathic effect(CPE) due to adenovirus infection. Cultures were examined daily for CPE,and the day upon which each experimental culture showed 100% CPE wasnoted. Reference 293 cell cultures infected with a range of knownamounts of adenovirus type-2 (from 0 to 1×10⁷ plaque forming units(pfu)/culture) were also prepared and treated in the same manner. Astandard curve was then prepared from the data obtained from thereference cultures, where the adenovirus pfu number was plotted againstthe day of 100% CPE. The titer of infectious adenovirus type-2 in eachexperimental culture was then readily obtained as determined from thestandard curve. The limit of detection of the assay was 100 pfu/mL. Thepresence of wild-type AAV contamination, analyzed by dot-blothybridization, was approximately 7 logs lower than recombinant virionconcentration.

[0144] Differentiation of Myoblasts

[0145] C2C12 myoblasts were transduced either while actively dividing,or as a differentiated cell culture. Differentiation was induced byplacing subconfluent myoblasts in murine differentiation medium (DMEMcontaining 2% horse serum and standard concentrations of glutamine andpenicillin-streptomycin) for an interval of four days prior totransduction in order to induce myoblast fusion and formation ofdifferentiated myotubes.

[0146] Fetal human skeletal myoblasts were differentiated in humandifferentiation medium (DMEM containing 10% horse serum and 5 μg/mLgentamicin). Verification of differentiation was performed bymicroscopic analysis to determine the presence of multinucleatedmyotubes in culture.

EXAMPLE 1 Expression of rAAV-LacZ in Terminally Differentiated Adult RatCardiomyocytes

[0147] The ability of recombinant AAV virions to transduce terminallydifferentiated adult cardiomyocytes was established in vitro.Cardiomyocytes were harvested by coronary perfusion with collagenase ofadult rat hearts (Fischer 344, Harlan Sprague Dawley, Indianapolis,Ind.). Cardiomyocytes were grown on laminin-coated glass coverslips andexposed to rAAV-LacZ virions for 4 hours. After 72 hours, the cells werestained for β-galactosidase activity. AAV expression was detected byblue staining of the binucleated cells. These studies demonstrated theability of rAAV virions to transduce terminally differentiated cells.The transduction efficiency in vitro was 30% of adult cells at amultiplicity of infection (MOI) of 10⁴ genomes per cell.

EXAMPLE 2 Stability of rAAV-LacZ Expression In Vivo

[0148] Adult Fischer rats were used to analyze expression of transgenesin vivo. Incremental doses of rAAV-LacZ virions were injected into theleft ventricular apex of the heart using either a subxyphoid or lateralthoracotomy approach. More particularly, experimental animals wereanesthetized with Metofane followed by a subxyphoid incision to exposethe diaphragmatic surface of the heart. Apical cardiac injections wereperformed with a glass micropipette. Recombinant virion was diluted innormal saline and injected at a volume of 20-50 μL.

[0149] At varying times post-injection, hearts were harvested andexamined for β-galactosidase production and for the presence ofinfiltrating mononuclear cells. For 5-Bromo-4-chloro-3-indolylβ-D-galactoside histochemical determination, frozen sections (6 μm) werefixed in 0.5% glutaraldehyde and stained for β-galactosidase activity asdescribed (Sanes et al. (1986) “Use of Recombinant Retrovirus to StudyPost-Implantation Cell Lineage in Mouse Embryos,” EMBO J 5:3133-3142).Paraffin sections (5 μm) were stained with hematoxylin/eosin. Sectionswere examined for infiltrating mononuclear cells.

[0150] The above-described histochemical studies showed greater than 50%transduction of cardiomyocytes in the region of injection at each timepoint examined. Further, there was no inflammatory cell infiltrate notedduring the course of analysis. β-galactosidase staining was observed topersist in cardiac muscle for at least two months following genetransfer.

EXAMPLE 3 In Vivo Transduction of Murine Skeletal Muscle using rAAV-LacZVirions

[0151] Recombinant AAV-LacZ virions were injected into muscle tissue ofmice, and transduction assessed by β-gal activity. Particularly, in vivotransduction was performed by intramuscular (IM) injection ofrecombinant AAV virions into the skeletal muscle of healthy 6-8 week oldBalb/c mice (Jackson Laboratories, Bar Harbor, Me., SimonsenLaboratories, Gilroy, Calif., or Harlan Laboratories) under eitherMetofane (Pitman-Moore, Mundelein, Ill.) or ketamine-xylazineanesthesia. The mid-portion of each tibialis anterior muscle was exposedvia a 1 cm incision. Injections into the tibialis anterior were carriedout using a micro-capillary tube attached to a Hamilton syringe toadminister the following formulations at a depth of 2 mm:phosphate-buffered saline (PBS) alone (negative control); or PBScontaining either rAAV-LACZ virions or pW1909adhlacZ plasmids.

[0152] Tissue samples of tibialis anterior muscle, forelimb muscle,heart, brain and liver were obtained for analysis of β-galactosidaseexpression. One tibialis anterior muscle from each animal was processedfor cross-sectional β-galactosidase analysis, and total β-galactosidasewas determined from a crude homogenate of the other muscle using achemiluminescent assay.

[0153] For histochemical detection of β-galactosidase, muscle sampleswere snap-frozen in dry ice-cooled isopentane, followed by serialtransverse sectioning (10 μm) and processing according to previouslydescribed methods (Sanes et al. (1986) EMBO J 5:3133-3142). Thecross-sectional area of the tibialis anterior expressing β-galactosidasewas determined as follows: after counter-staining with nuclear fast red(Vector Labs), the X-gal stained tissue was digitally photographed andthe cross-sectional area of stored images was determined using NIH Imagesoftware.

[0154] The GALACTO-LIGHT™ (Tropix, Bedford, Mass.) chemiluminescentreporter assay kit was used to detect total β-galactosidase activity inthe entire tibialis anterior muscle. Standard curves were prepared fromknown amounts of purified β-galactosidase (Sigma, St. Louis, Mo.)resuspended in non-transduced muscle homogenate. β-galactosidaseactivity is expressed as either: nanograms of β-galactosidase,normalized for the entire muscle, minus background activity; or in termsof relative light units (RLU) as quantified by luminometer. Forelimbmuscle, cardiac muscle, brain tissue and liver samples were assayed inan identical fashion.

[0155] A. Time Course of β-Galactosidase Expression.

[0156] A single intramuscular injection into the left and right tibialisanterior muscles (under direct vision) was used to deliver 8×10⁹rAAV-LacZ in a PBS vehicle. Four animals were injected with PBS alone.Animals were sacrificed at 2, 4, 8, 12, 24 and 32 weeks after injection,and the tibialis anterior muscle was excised and analyzed for thepresence of bacterial β-galactosidase (n=5 for each group) as describedabove.

[0157] Efficiency of the LacZ gene transfer was assessed bycross-sectional tissue staining and chemiluminescent assay. As can beseen in FIG. 1 and in Table I below, gene expression persisted for atleast 32 weeks. In addition, two weeks after injection of therecombinant virions, 18% of the muscle cross-sectional area expressedβ-galactosidase, while at 32 weeks, 24% of the muscle cross-sectionalarea expressed β-galactosidase. Negative control tibialis anteriormuscle (obtained from the animals injected with PBS alone) showed nobackground staining. Muscle β-galactosidase activity was also determinedin the contralateral injected muscle. This study also revealedpersistent expression for at least 32 weeks, in agreement with thecross-sectional fiber analysis (Table I). Further, the observedβ-galactosidase was sustained with minimal inflammatory cell infiltrate.These data demonstrate that rAAV-LacZ virion administration into muscleresults in stable expression of the transgene for at least 8 months.

[0158] Upon histological examination, positive staining filled thecytoplasm of the transduced myofibers and was observed through largecontiguous portions of the, muscle. Serial transverse sections revealedthat blue staining extended throughout the length of the muscle fiber.Diffraction-interference contrast microscopy revealed a cleardelineation between positively and negatively stained myofibers (FIG.2), suggesting that recombinant virion delivery was limited bystructural barriers such as epimyseal or perimyseal connective tissue.Homogenates prepared from brain, heart, liver, and forelimb muscledisplayed no β-galactosidase activity when compared with the backgroundactivity of the negative control animals. TABLE I Time Course ofβ-galactosidase Expression Following Single Injection with rAAV-LacZ.Percent cross- β-galactosidase sectional area Time expression expressingβ- (weeks) (ng/muscle) galactosidase (%) 2 1441 ± 458  18 ± 6  4 951 ±176 20 ± 4  8 839 ± 436 24 ± 3  12 1878 ± 521  29 ± 5  24 2579 ± 1165 22± 3  32 1242 ± 484  24 ± 3  # for histochemical detection ofβ-galactosidase and determination of the cross-sectional area of thetibialis anterior expressing β-galactosidase. Mean cross-sectional areas± SEM are shown.

[0159] B. Dose-Response Assay.

[0160] To determine the effective dose range for rAAV-LacZ in vivo,recombinant virions were injected into the tibialis anterior muscle of6-8 week old Balb/c mice, and transduction assessed by β-galactosidaseactivity as measured by GLACTO-LIGHT™ relative light units (RLU). As canbe seen in FIG. 3, at two weeks post-injection, the observed RLU rangedfrom approximately 0.2×10⁷ RLU/muscle (injected with 8×10⁸ rAAV-LacZ) toapproximately 1.1×10⁹ RLU/muscle (injected with 3.6×10¹¹ rAAV-LacZ).

[0161] The levels of expression of β-galactosidase measured in RLUcorrespond to the percentage of β-galactosidase-positive muscle fiberson cross-sectional analysis. For example, 0.2×10⁷ RLU corresponds toapproximately 1% β-galactosidase positive muscle fibers and 1.1×10⁹ RLUcorresponds to approximately 60% β-galactosidase positive muscle fibers.

[0162] C. Comparison of β-Galactosidase Expression Efficiency.

[0163] A comparison of β-galactosidase expression efficiency obtained byin vivo transduction of mice using either rAAV-LacZ virions, or plasmidDNA containing the same LacZ expression cassette (pW1909adhlacZ) wascarried out as follows. Either 8×10⁹ rAAV-LacZ, or 100 μg ofpW1909adhlacZ, was injected into the tibialis anterior muscle of 6-8week old Balb/c mice. Two weeks post-injection, β-galactosidase activitywas assessed using the GALACTO-LIGHT™ chemiluminescent reporter assaykit, as described above. Administration of the recombinant virionsresulted in 1441 ng β-galactosidase/muscle (n=5), while administrationof 100 μg of the plasmid DNA, a typical in vivo plasmid DNA dosage(Whalen et al. (1995) Hum. Gene Ther. 4:151-159), resulted in 12 ngβ-galactosidase/muscle (n=4). This dosage of pW1909adhlacZ plasmid DNAis equivalent to 2.2×10¹³ single stranded genomes, demonstrating thatgene delivery by the recombinant virions was substantially moreefficient than delivery of an equal molar quantity of vector DNA.

EXAMPLE 41 In Vitro Transduction of Murine Myotubes and Myoblasts

[0164] In order to determine if differentiated cultured muscle cells areappropriate targets for recombinant AAV virion transduction, and toassess the ability of such cells to express a transduced gene, thefollowing study was carried out. Murine C2C12 cells were selected sincethese cells have been extensively studied as a model for mammalianmyogenesis (Blau et al. (1993) Trends Genet. 9:269-274), and can beinduced to differentiate by growth in reduced serum medium.

[0165] In the study, C2C12 myoblasts (dividing cells) were seeded incell culture plates at a density of 2×10⁴ cells/cm², maintained ingrowth media (GM) until confluent, split, and then either cultured in GMor cultured for 5 days in murine DM. Differentiation was verified by themicroscopic presence of multinucleate myotubes, representing fusedmyoblasts (differentiated C2Cl2 cells).

[0166] The C2C12 myotubes and myoblasts were transduced in culture withpurified rAAV-hEPO virions at a MOI of 10⁵ in OptiMEM (Gibco BRL). Inthe myotube cultures, DM was added after virion adsorption. The culturemedia of the transduced cells was changed 24 hours prior to collectionof supernatants at 3, 8 and 14 days following transduction. Secretion ofhEPO was assessed by ELISA using the human erythropoietin Quantikine IVDkit (available from R and D Systems, Minneapolis, Minn.) according tomanufacturer's recommendations.

[0167] The results of the study show that hEPO is secreted from both thetransduced myotubes and myoblasts. The levels of hEPO secretionincreased in the myotubes over the first seven days post-transduction(FIG. 4). As can be seen by reference to FIG. 5, a dose-dependentincrease in the secretion of hEPO was also observed in the transducedC2C12 myotubes. Eight days post-transduction of the myotubes, hEPOlevels peaked at >3400 mU/mL. These data demonstrate that transductionwith rAAV-hEPO of both myotubes or myoblasts results in hEPO secretionby the transduced cells, and that in short-term myotube cultures, hEPOis synthesized and secreted in a dose-dependent manner.

EXAMPLE 5 In Vitro Transduction of Human Myotubes Using rAAV-hEPOVirions

[0168] To determine if differentiated primary human muscle cells areable to express hEPO following transduction with rAAV-hEPO, thefollowing study was carried out. Primary fetal human skeletal myoblastswere seeded in cell culture plates at a density of 2×10⁴ cells/cm²,grown to confluence in appropriate growth media, and then cultured for14 days in human DM. Differentiation was verified by microscopicexamination for multinucleate cells. In vitro transduction was carriedout by adding purified rAAV-hEPO virions to the cultured myotubes inOptiMEM medium (Gibco BRL). DM was added to the cultures after virionadsorption. Control cultures were transduced with rAAV-LacZ.

[0169] Culture media was changed 24 hours prior to collection ofsupernatants at day 3, 8 and 14 post transduction. Secreted EPO levelswere assayed by ELISA as described above in Example 4.

[0170] As can be seen in FIG. 6, the transduced human myotubes secretedhEPO into the culture in a dose-dependent manner. No detectable EPOactivity was measured in the control cultures. Secretion of EPOincreased over the 14-day interval post-transduction. These datademonstrate that primary human myotubes transduced by recombinant AAVvirions are capable of expressing and secreting erythropoietin.

EXAMPLE 6 Systemic Delivery of Human Erythropoietin In Vivo byIntramuscular Administration of rAAV-hEPO

[0171] Recombinant AAV virions encoding hEPO were administered to adulthealthy Balb/c mice in vivo to determine if a systemic level of hEPO canbe produced, and a biological response obtained. At various time pointsafter administration, blood was obtained from the orbital venous plexusunder anesthesia. Serum hEPO levels were determined by ELISA asdescribed above. Red cell counts were done by hemocytometer, hematocritwas determined by centrifugation of blood in micro-capillary tubes, andhemoglobin concentration was analyzed by cyanmethemoglobin assay (DMA,Arlington, Tex.) according to manufacturer's specifications and comparedwith a standard (Stanbio Laboratory, San Antonio, Tex.) analyzed at 570nm on a spectrophotometer. Reticulocytes were analyzed by either newmethylene blue stain, or by FACS analysis of thiazole orange stainedperipheral blood samples (RETIC-COUNT®, Becton-Dickinson, Mountain View,Calif.); the results of data obtained by either of these methods weresimilar.

[0172] An initial experiment revealed that high levels of hEPO andelevated hematocrits were maintained for >100 days in mice injected IMwith 6.5×10¹¹ rAAV-hEPO. Next, adult female Balb/c mice were injected IMin both hind limbs with a single administration of rAAV-hEPO at dosagesranging from 3×10⁹ to 3×10¹¹ particles. Control animals were injectedwith rAAV-LacZ. The resulting serum hEPO levels were analyzed and arereported below in Table II. As can be seen, a well-defined dose-responsewas obtained 20, 41, 62 and 83 days post injection.

[0173] The time course of hEPO secretion by animals receiving rAAV-hEPOis depicted in FIG. 7. As can be seen, serum levels of hEPO increasedwith time to plateau at from 6 to 8 weeks after injection.

[0174] The biological activity of secreted hEPO can be monitored byelevation of hematocrit in the experimental animals. A comparison ofcirculating hEPO levels versus hematocrit is shown in Table II. Thecomparison shows that hematocrit increased with time and increasingrecombinant virion dose. Further, stable elevation in hematocrit hasbeen observed for up to 40 weeks in a group of experimental animalsinjected with rAAV-hEPO. Control animals had undetectable levels of hEPO(<2.5 mU/mL, the lower limit of detection for the assay).

[0175] These results indicate that persistent and stable high-levelsecretion of hEPO, with a corresponding elevation in hematocrit, isestablished following a single IM administration of-rAAV-hEPO.

[0176] In addition, comparison of the expression of hEPO by animalsinjected IM with rAAV-hEPO (3×10¹¹ single-stranded genomes) and animalsinjected IM with the pW1909EPO plasmid (1.4×10¹³ double-stranded genomesin 100 μg DNA) shows that the recombinant virions gave rise tosignificantly greater levels of EPO expression. As reported in Table II,20 days post-injection, recombinant virion-injected animals had serumlevels of 445±98 mU/mL, while the plasmid-injected animals had levels of8±10 mU/mL. At 41 days post-injection, levels in the recombinantvirion-treated animals had risen to 725±112 mU/mL, while the levels inthe plasmid-treated animals had dropped below the level of detection.The animals receiving rAAV-hEPO exhibited approximately 60-fold morecirculating hEPO with 100-fold less input genomes at 20 dayspost-injection, or approximately 6000-fold greater secretion per genome.At 41 days post-injection, this difference was even greater, since theplasmid expression was below the level of detection. TABLE II EPOExpression and Hematocrit: rAAV-hEPO Dose-Response Days afterAdministration 20 days 41 days 62 days 83 days Dose EPO HCT EPO HCT EPOHCT EPO HCT 3 × 10¹¹ 445 ± 98  74.2 ± 1.2  725 ± 112 82.3 ± 1.2  769 ±61  86.5 ± 1.4  723 ± 253 88.5 ± 0.7  1 × 10¹¹ 85 ± 14 72.8 ± 1.5  212 ±23    79.5 ± 1.7  234 ± 75  83.2 ± 0.2  220 ± 51  83.2 ± 2   3 × 10¹⁰ 17± 5  60.0 ± 3.5  34 ± 17   74.7 ± 3.2  55 ± 28 78.7 ± 2.0  73 ± 45 80.0± 3   1 × 10¹⁰ 3 ± 1 52.9 ± 1.8  11 ± 3    61.5 ± 1.9  12 ± 8  68.4 ±4.6  15 ± 5  70.8 ± 8   3 × 10⁹ <2.5 49.9 ± 1.4  <2.5   53.5 ± 2.5  <2.557.0 ± 2.4  4 ± 4 57.5 ± 3   i.v. 7 ± 3 54.7 ± 3.2   13 ± 2.0 <64.4 ±5.3   10.1 ± 0.7  70.8 ± 8   21 ± 10 74.6 ± 7   Control <2.5 48.9 ± 1.0 <2.5   49.1 ± 0.8  <2.5 48.1 ± 0.7  <2.5 48.2 ± 9   Plasmid  8 ± 10  50± 3.0 <2.5   50.2 ± 1.0  <2.5 47.8 ± 0.9  N.D. N.D.

EXAMPLE 7 A Comparison of hEPO Secretion from rAAV-hEPO Administered byIM or IV Routes

[0177] A comparison of the circulating levels of hEPO resulting from IMand IV routes of administration was analyzed to determine which methodof gene delivery results in higher levels of systemic hEPO. Balb/c micewere injected with 3×10¹¹ rAAV-hEPO using either the IM route asdescribed above, or intravenously (IV) in PBS in a total volume of 50 μLvia the lateral tail vein. Serum hEPO levels were determined by ELISAusing the methods described above.

[0178] As shown in Table II, HEPO levels resulting from the IVadministrations were significantly lower than the group that receivedthe virions by the IM route. In particular, at 20 days post-injection,the IM route resulted in levels of hEPO of 445±98 mU/mL, while the IVroute produced 7±3.0 mU/mL. At 41 days post-injection, the EPO levelobserved with the IM route was 725±112 as compared with 13±2.0 mU/mL byIV, or approximately 60-fold more efficacious. These data demonstratethat the IM route of injection resulted in higher systemic levels ofhEPO, and suggest that interstitial delivery in muscle results inimproved transduction by the recombinant AAV virions.

EXAMPLE 8 In Vitro and In Vivo Transduction of Muscle Cells UsingrAAV-GAA Virions

[0179] Cardiomyopathy in infancy is frequently due to inheritedmetabolic disease. One such metabolic disease, glycogen storage diseasetype II (Pompe's disease) is an inherited cardiomyopathy caused by adeficiency in the lysosomal enzyme, acid α-glucosidase (GAA). GAAfunctions to cleave α-1,4 and α-1,6 linkages of lysosomal glycogen torelease monosaccharides. Loss of enzyme activity results in accumulationof lysosomal glycogen in striated muscle, and is characterized bylysosomal rupture, contractile apparatus disruption and glycogeninfiltration. Currently, no effective treatment is available.

[0180] Accordingly, the following studies were carried out to determinewhether the recombinant AAV virions of the present invention can be usedto obtain long-term expression of GAA in transduced muscle cells.

[0181] A. In Vitro Transduction of Human Skeletal Muscle Cells withrAAV-hGAA Virions.

[0182] Human skeletal myoblasts were seeded in cell culture plates at adensity of 2×10⁴ cells/cm², grown to confluence in appropriate growthmedia, and then cultured for 14 days in human DM. Differentiation wasverified by microscopic examination for multinucleate cells. In vitrotransduction was carried out by adding purified rAAV-hGAA virions at anMOI of 2×10⁵ to the cultured myotubes in OptiMEM medium (Gibco BRL). DMwas added to the cultures after virion adsorption. Transduced controlcultures were established by transducing the myotubes with rAAV-LacZ atthe same MOI, and negative controls were established by culturingnon-transduced myotubes.

[0183] Culture media was changed 24 hours prior to collection ofsupernatants at day 3, 8 and 14 post transduction. hGAA expression wasdetermined by enzymatic assay. Specifically, cell monolayers wereharvested with 0.05% trypsin in Puck's saline A containing 0.02%ethylenediaminetetraacetic acid. After quenching the trypsin with growthmedia, cells were centrifuged, the cell pellet washed with PBS, andresuspended in distilled water. Following three freeze-thaw cycles, thesamples were microfuged at 10,000×g. Protein in the supernatant wasdetermined using the Bicinchoninic acid method (Pierce) with bovineserum albumin (BSA) as the standard. GAA activity was measured usingcleavage of the glycogen analog 4-methylumbelliferyl-α-D-glucoside, by amodification of a previously described method (Galjaard et al. (1973)Clin. Chim. Acta. 49:361-375; Galjaard, H. (1973) Pediatr. Res. 7:56).Assays contained 30 μg protein in 125 μL water. Two volumes of 200 mMsodium acetate (pH 4.3) and 750 nM 4-methyumbelliferyl-α-D-glucoside(from a stock solution of 200 mM in dimethylsulfoxide) were added andthe samples incubated at 37° C. for one hour. Reactions were stoppedwith 625 μL sodium carbonate (pH 10.7). Once cleaved and alkalinized,the 4-methyumbelliferyl fluoresces. Measurements were made with afluorescence spectrophotometer with excitation at 365 nm and emission at448 nm. Assay measurements were compared against 4-methyumbelliferone(Sigma, St. Louis, Mo.) standards. Zero protein and zero time blankswere used.

[0184] The observed GAA activity is reported in FIG. 8 whichdemonstrates that in vitro transduction of the human myotubes results inhigh level GAA expression at day 8 and 14 post transduction.

[0185] B. In Vivo Transduction of Skeletal Muscle Using rAAV-GAAVirions.

[0186] Recombinant AAV virions encoding human GAA were administered toadult Balb/c mice in vivo to determine if systemic levels of GAA can beproduced by the transduced cells. Muscle tissue was isolated at varioustime points after administration, and processed for GAA activity usingan enzymatic assay.

[0187] In the study, tibialis anterior muscle in Balb/c mice wassurgically exposed, and a single intramuscular injection into the leftand right muscles (under direct vision) was used to administer thefollowing formulations: phosphate-buffered saline (PBS) alone (negativecontrol); or PBS containing either 2×10¹⁰ rAAV-hGAA or rAAV-LacZ (for atotal of 4×10¹⁰ virions/animal).

[0188] Tissue samples of tibialis anterior muscle were obtained at 1, 4and 10 weeks following transduction. The tissue samples were prepared byhomogenization in water followed by three freeze-thaw cycles.Freeze-thaw lysates were microcentrifuged, and the resultant supernatantassayed for GAA activity as described above. The results of the studyare depicted in FIG. 9. As can be seen, stable expression of GAA in thetransduced mouse muscle cells was observed for ten weeks, demonstratingthat the recombinant AAV virions of the present invention are able toestablish efficient expression of a functional lysosomal protein intransduced cells, and thus provide a therapeutic approach for thetreatment of glycogen storage disease.

[0189] Accordingly, novel methods for transferring genes to muscle cellshave been described. Although preferred embodiments of the subjectinvention have been described in some detail, it is understood thatobvious variations can be made without departing-from the spirit and thescope of the invention as defined by the appended claims.

[0190] Deposits of Strains Useful in Practicing the Invention

[0191] A deposit of biologically pure cultures of the following strainwas made with the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md., under the provisions of the Budapest Treaty. Theaccession number indicated was assigned after successful viabilitytesting, and the requisite fees were paid. Access to said cultures willbe available during pendency of the patent application to one determinedby the Commissioner to be entitled thereto under 37 CFR 1.14 and 35 USC122. All restriction on availability of said cultures to the public willbe irrevocably removed upon the granting of a patent based upon theapplication. Moreover, the designated deposits will be maintained for aperiod of thirty (30) years from the date of deposit, or for five (5)years after the last request for the deposit; or for the enforceablelife of the U.S. patent, whichever is longer. Should a culture becomenonviable or be inadvertently destroyed, or, in the case ofplasmid-containing strains, lose its plasmid, it will be replaced with aviable culture(s) of the same taxonomic description.

[0192] This deposit is provided merely as a convenience to those ofskill in the art, and is not an admission that a deposit is required. Alicense may be required to make, use, or sell the deposited materials,and no such license is hereby granted. Strain Deposit Date ATCC No.pGN1909 Jul. 20, 1995 69871

[0193]

1 9 14 base pairs nucleic acid single linear DNA (genomic) 1 GGCAGCTGCCTGCA 14 14 base pairs nucleic acid single linear DNA (genomic) 2GAAGGCGCGC CTTC 14 80 base pairs nucleic acid single linear DNA(genomic) 3 GCGGCCGCAC GCGTACGTAC CGGTTCGAAG CGCGCACGGC CGACCATGGTTAACTCCGGA 60 CACGTGCGGA CCGCGGCCGC 80 28 base pairs nucleic acid singlelinear DNA (genomic) 4 AAAATTCGAA CCTGGGGAGA AACCAGAG 28 28 base pairsnucleic acid single linear DNA (genomic) 5 GTTTCCCCGC GAATGGACAAGCTTAAAA 28 91 base pairs nucleic acid single linear DNA (genomic) 6GCGGCCGCAC GCGTTGTTAA CAACCGGTTC GAAGCGCGCA GCGGCCGACC ATGGGTTTAA 60ACTCCGGACC ACGTGCGGAC CGAGCGGCCG C 91 28 base pairs nucleic acid singlelinear DNA (genomic) 7 AAAATTCGAA CAGGTAAGCG CCCCTTTG 28 28 base pairsnucleic acid single linear DNA (genomic) 8 GAGACCAAAG AGGGGTCCAAGCTTAAAA 28 101 base pairs nucleic acid single linear DNA (genomic) 9GAGCTCAATC GATTGAATTC CCCGGGGATC CTCTAGAGTC GACCTGCAGC CACTGTGTTG 60GATATCCAAC ACACTGGTAG GGATAACAGG GTAATCTCGA G 101

1. A method of delivering a selected gene to a muscle cell or tissue,said method comprising: (a) providing a recombinant adeno-associatedvirus (AAV) virion which comprises an AAV vector, said AAV vectorcomprising said selected gene operably linked to control elementscapable of directing the in vivo transcription and translation of saidselected gene; and (b) introducing said recombinant AAV virion into saidmuscle cell or tissue.
 2. The method of claim 1, wherein said musclecell or tissue is derived from skeletal muscle.
 3. The method of claim1, wherein said muscle cell or tissue is derived from smooth muscle. 4.The method of claim 1, wherein said muscle cell or tissue is derivedfrom cardiac muscle.
 5. The method of claim 1, wherein said muscle cellis a skeletal myoblast.
 6. The method of claim 1, wherein said musclecell is a skeletal myocyte.
 7. The method of claim 1, wherein saidmuscle cell is a cardiomyocyte.
 8. The method of claim 1, wherein saidrecombinant AAV virion is introduced into said muscle cell in vivo. 9.The method of claim 8, wherein said recombinant AAV virion is introducedby intramuscular injection.
 10. The method of claim 1, wherein saidrecombinant AAV virion is introduced into said muscle cell in vitro. 11.The method of claim 1, wherein said selected gene encodes a therapeuticprotein.
 12. The method of claim 11, wherein said protein is acidα-glucosidase.
 13. A muscle cell or tissue transduced with a recombinantAAV virion which comprises an AAV vector, said AAV vector comprising aselected gene operably linked to control elements capable of directingthe in vivo transcription and translation of said selected gene.
 14. Themuscle cell of claim 13, wherein said cell is a skeletal myoblast. 15.The muscle cell of claim 13, wherein said cell is a skeletal myocyte.16. The muscle cell of claim 13, wherein said cell is a cardiomyocyte.17. The muscle cell of claim 13, wherein said selected gene encodes atherapeutic protein.
 18. The muscle cell of claim 17, wherein saidselected gene encodes acid α-glucosidase.
 19. A method of treating anacquired or inherited disease in a mammalian subject comprisingintroducing into a muscle cell or tissue of said subject atherapeutically effective amount of a pharmaceutical composition whichcomprises (a) a pharmaceutically acceptable excipient; and (b)recombinant AAV virions, wherein said recombinant AAV virions comprisean AAV vector, said AAV vector comprising a selected gene operablylinked to control elements capable of directing the transcription andtranslation of said selected gene when present in said subject, whereinsaid introducing is done in vivo.
 20. A method of treating an acquiredor inherited disease in a mammalian subject comprising: (a) introducinga recombinant AAV virion into a muscle cell or tissue in vitro toproduce a transduced muscle cell, wherein said recombinant AAV virioncomprises an AAV vector, said AAV vector comprising a selected geneoperably linked to control elements capable of directing thetranscription and translation of said selected gene when present in saidsubject; and (b) administering to said subject a therapeuticallyeffective amount of a composition comprising a pharmaceuticallyacceptable excipient and the transduced muscle cells from step (a). 21.A method for delivering a therapeutically effective amount of a proteinsystemically to a mammalian subject comprising introducing into a musclecell or tissue of said subject a pharmaceutical composition whichcomprises (a) a pharmaceutically acceptable excipient; and (b)recombinant AAV virions, wherein said recombinant AAV virions comprisean AAV vector, said AAV vector comprising a selected gene operablylinked to control elements capable of directing the transcription andtranslation of said selected gene when present in said subject, whereinsaid introducing is done in vivo.
 22. A method for delivering atherapeutically effective amount of a protein systemically to amammalian subject comprising: (a) introducing a recombinant AAV virioninto a muscle cell or tissue in vitro to produce a transduced musclecell, wherein said recombinant AAV virion comprises an AAV vector, saidAAV vector comprising a selected gene operably linked to controlelements capable of directing the transcription and translation of saidselected gene when present in said subject; and (b) administering tosaid subject a therapeutically effective amount of a compositioncomprising a pharmaceutically acceptable excipient and the transducedmuscle cells from step (a).
 23. An adeno-associated virus (AAV) vectorcomprising a gene encoding acid α-glucosidase operably linked to controlelements capable of directing the in vivo transcription and translationof said gene.
 24. A recombinant adeno-associated virus (AAV) virionwhich comprises the AAV vector of claim 23.